Prevalence of phenotypic multi-drug resistant Klebsiella species recovered from different human specimens in Ethiopia: A systematic review and meta-analysis

Background Multidrug-resistant (MDR) Klebsiella species are among public health important bacteria that cause infections difficult to treat with available antimicrobial agents. Infections with Klebsiella lead to high morbidity and mortality in developing countries particularly in patients admitted to the intensive care unit. This systematic review and meta-analysis aimed to determine the pooled prevalence of MDR Klebsiella species from different human specimens using studies conducted in Ethiopia from 2018−2022. Methods We have systematically searched online databases such as PubMed/Medline, Google Scholar, Hinari, African journals online, Web of Science, Cochrane, and grey literature (Addis Ababa University and Hawassa University) to identify studies reporting the proportion of MDR Klebsiella species in Ethiopia. Published articles were selected based on the Preferred Reporting Item of Systematic Review and Meta-analysis (PRISMA). R-Studio version 4.2.3 was used to conduct pooled prevalence, heterogeneity test, and publication bias. A binary random effect model was used to determine the pooled prevalence. Heterogeneity was checked with the inconsistency index (I2). Publication bias was checked with a funnel plot and Egger test. Sensitivity analysis was conducted with leave-one-out analysis. Joanna Briggs Institute’s critical appraisal tool for prevalence studies was used to check the quality of each article. Results In this systematic review and meta-analysis, 40 articles were included in which 12,239 human specimens were examined. Out of the total specimens examined, 721 Klebsiella species were isolated and 545 isolates were reported to be MDR Klebsiella species. The prevalence of MDR Klebsiella species ranged from 7.3%-100% whereas the pooled prevalence of MDR Klebsiella species was 72% (95% CI: 63 − 82%, I2 = 95%). Sub-group analysis based on region revealed the highest prevalence of MDR from Addis Ababa (97%) and the least from the Somali region (33%); whereas sub-group analysis based on the specimen type indicated the highest prevalence was from blood culture specimens 96% and the least was from other specimens (ear and vaginal discharge, and stool) (51%). Conclusion Our finding indicated a high prevalence of MDR Klebsiella species found in different human specimens. The prevalence of MDR Klebsiella varies across regions in Ethiopia, age, the type of specimens, source and site of infection. Therefore, integrated action should be taken to reduce the prevalence of MDR Klebsiella species in regional states and focus on clinical features. Effective infection and prevention control should be applied to reduce the transmission within and outside health care settings.


Introduction
Klebsiella is a member of the Enterobacteriaceae family and is naturally present in the gastrointestinal tract microbiome of healthy humans and animals [1].This common pathogen causes hospital-acquired surgical wound infections, digestive tract infections, community-onset infections, and nosocomial infection outbreaks [2].
Genus Klebsiella includes a variety of species that cause human such as Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca and Klebsiella aerogenes [3].The two main pathotypes of Klebsiella species, the multidrug-resistant (MDR) and hypervirulent (hv) clones, account for a significant fraction of infections [4].The two branches' strains were regarded as non-overlapping since their respective genetic backgrounds differed [4].Nevertheless, it has been shown that Klebsiella species.can obtain genetic elements and mutations that confer virulence traits and/or antibiotic resistance, which ultimately leads to the emergence of convergent clones known as multidrug-resistant and hyper-virulent (MDRhv) Klebsiella spp [5,6].MDR-hv Klebsiella species exhibit dual hyper-virulence and antibiotic resistance, and they are believed to be evolving further to generate phenotypically distinct strains [6,7].Numerous reports from various continents worldwide have documented a wide range of MDR-hv strains of Klebsiella species that have evolved through various mechanisms [8].MDR-hv Klebsiella species have become real superbugs that pose major threats to public health due to the rise in severe infections and the growing lack of effective treatments [9].
In the past decade, antibiotic resistance has become a major global public health concern.Antimicrobial resistance (AMR)-related infections caused 700,000 deaths worldwide each year; by 2050, this number is predicted to increase to 10 million worldwide and 4.2 million in Africa [10].According to reports, there is a 70% global rate of antibiotic resistance for Klebsiella, and the incidence of infection-related mortality ranges from 40% to 70% [4].In recent years, there has been a growing concern for global public health regarding carbapenem-resistant Klebsiella (CRK) and multiple-drug-resistant Klebsiella (MDRK).Klebsiella species was listed as one of the eight drug-resistant microorganisms in a 2017 WHO report.The advent of multidrug-resistant Klebsiella species and limited therapeutic options for infections caused by multidrug-resistant Klebsiella species has made treating infections caused by these species more difficult at the moment [11,12].
Even though there is a clear correlation between colonization and infection, it is unknown what risk factors colonized patients have for infection.It is most likely determined by a combination of bacterial and patient factors whether a patient becomes infected with Klebsiella.In a population-level analysis lacking colonization assessment, Klebsiella bacteremia was linked to advanced age, male sex, dialysis, chronic liver disease, solid organ transplant, and cancer [13].Advanced age is linked to the colonization of Klebsiella [14].Klebsiella genes and patient characteristics linked to infection with Klebsiella as opposed to asymptomatic colonization [15].
Due to their multidrug resistance, Klebsiella species is now considered an urgent threat to human health; however, the MDR profile varies amongst countries, even when it comes to the widespread use and misuse of antimicrobial agents [16].The epidemiology and drug susceptibility pattern of Klebsiella species have been studied in various regions of Ethiopia at different times, but the pooled prevalence of MDR Klebsiella species in Ethiopia has not been reported.This systematic review and meta-analysis aimed to determine the pooled prevalence of MDR Klebsiella species in Ethiopia based on regional states, specimen types, age, source and site of infections.

Literature search strategy
We have systematically searched online databases such as PubMed/Medline, Google Scholar, Hinari, African journals online, Web of Science, Cochrane, and grey literature (Addis Ababa University and Hawassa University) to identify studies reporting the proportion of multi-drug resistant Klebsiella species in Ethiopia.Search terms were grouped into four queries (prevalence, antimicrobial susceptibility related term, Klebsiella spp, and Ethiopia).The Boolean operator, 'or' (within a query) 'or' (between antibacterial resistance terms), Resistance search terms include "Antibiotic-resistant Klebsiella", "Antibiotic susceptible Klebsiella", "Klebsiella antibiotic sensitivity", "Antimicrobial susceptibility of Klebsiella", "Antimicrobial sensitivity of Klebsiella", "Antimicrobial resistance of Klebsiella", "Antibacterial resistance of Klebsiella", "Resistance of Klebsiella", "Drug resistance of Klebsiella".Studies published from January 1, 2018, to March 3, 2023, were included.We followed the Preferred Reporting Item of Systematic Review and Meta-analysis (PRISMA) to select the articles [17] (S1 Table ).

Data extraction
The data were extracted by three researchers (BK, TA, and MMA) using a standardized and pretested format on April 1-7, 2023.The data abstraction format included the first author, study site, specimen, lab method, sample size, the number of Klebsiella species isolated and MDR profile of Klebsiella species.Any disagreement on the study to be included/excluded during data extraction between researchers was handled through discussion.

Quality assessment
Before considering the articles for systematic review and meta-analysis, their quality was checked using nine criteria mentioned in the Joanna Briggs Institute (JBI) critical appraisal tool for prevalence studies [18].The checklist includes the following questions: Was the sample frame appropriate to address the target population?Were study participants sampled appropriately?Was the sample size adequate?Were the study subjects and the setting described in detail?Was the data analysis conducted with sufficient coverage of the identified sample?Were valid methods used for the identification of the condition?Was the condition measured in a standard, reliable way for all participants?Was there an appropriate statistical analysis?Concerning articles based on specimen type, most of the articles (21 articles) reported MDR Klebsiella from urine [21-23, 25, 27, 30, 33, 38-41, 43, 44, 48-50, 53, 56, 58, 59].It is followed by seven (7) articles from blood [20,35,37,42,46,47,51], 4 articles from sputum [24,34,52,57], 3 articles from eye swabs [26,28,45], 2 articles from body fluid [31,55], 1 article for each of this specimen stool [32]: vaginal discharge [36] and ear discharge [29].All the susceptibility testing was conducted with the disk diffusion method.A total of 12,239 patient specimens were analyzed from this, 721 Klebsiella were isolated, and of this 545 were reported as MDR (Table 1).

Pooled prevalence of MDR Klebsiella species
A total of 12, 239 patients specimen were analyzed, from these 721 Klebsiella species were isolated, and 545 isolates were reported as MDR Klebsiella species.The review result indicates the prevalence ranged from 7.3%-100% (Table 1).According to meta-analysis, the overall pooled prevalence of MDR Klebsiella species in Ethiopia was 72% (63-82%) with high heterogeneity Because of statistically significant high heterogeneity, we have conducted subgroup analysis based on the regional state, specimen type, source of infection, age of study participants, and site of infection.

Meta-regression
Meta-regression was conducted using regional state, types of specimens, study participants' age, source of infection, site of infection, and publication year to check the source of the heterogeneity but no variables were significantly associated (Table 3).

Publication bias
The funnel plot's asymmetry suggests that there was publication bias, which is corroborated by Egger's test (P < 0.0001) (Fig 4).

Sensitivity analysis
During sensitivity analysis using leave-one-out analysis revealed that the pooled prevalence range of MDR Klebsiella remains unchanged (Table 4).

Discussion
In hospitalized patients, as well as those in nursing homes and other healthcare facilities, Klebsiella species can cause a range of infections, including bloodstream infections, pneumonia, and urinary tract infections more seriously in immunocompromised patients [60].Klebsiella are often resistant to multiple antibiotics.Evidence implicates plasmids as the primary source of the resistance genes.Klebsiella species with the ability to produce extended-spectrum betalactamases (ESBL) are resistant to virtually all beta-lactam antibiotics, except carbapenems [1].Developing countries are particularly severely affected by these resistant bacteria if appropriate action is not taken right away.Our study attempted to compile the dispersed data in Ethiopia using a systematic review and meta-analysis because it is one of the nations that has experienced improper antibiotic consumption.
In our finding, the pooled prevalence of MDR Klebsiella species was estimated to be 72% (63-82%).Our finding is higher than the study conducted at a global level estimates of MDR K. pneumoniae 32.8% [61] and Nepal 55% [62].This difference may be due to their study was conducted on only MDR K. pneumoniae from hospital-acquired infection isolates and our study included all Klebsiella species on both CAI and HAI.Our finding is comparable with systematic review and meta-analysis from Nepal 64% [63] which was conducted on MDR K. pneumoniae from clinical isolates even if our study is on overall Klebsiella species.It is also comparable with a study from Ethiopia 68% [64] which was conducted with one health approach from human, animal, and environmental samples.These similarities indicate MDR Klebsiella species is circulating anywhere and fighting AMR with one health approach is much more important nowadays.
Globally, AMR has been documented and has the potential to spread quickly.Along with the usage of antibiotics, there are differences in the distribution of resistance and the number of infections around the world.There is a significant knowledge gap regarding the magnitude of AMR in the world.Particularly the knowledge gap is common in low-and middle-income countries that lack systems to gather data on infections and antibiotic-resistant bacteria [65].In our systematic review and meta-analysis, a subgroup analysis was conducted based on the regional states, type of specimens, study participants' age, source of infection (CAI vs HAI), and types of infection as the data is heterogenous (I 2 = 95%).The MDR Klebsiella species prevalence varied among the regional states, according to the subgroup analysis.Addis Ababa had the highest pooled prevalence, at 97% (93-100%), while Somali had the lowest, at 33% (1-91%).The number of studies included the nature of study participants, and the sample size could all be contributing factors to this variation.One possible explanation could be variations in access to clean water, hygienic practices, and sanitation for both humans and animals; infection prevention and control measures at homes, hospitals, and farms; availability of reasonably priced vaccines; access to diagnostics and medications; and awareness and knowledge regarding antibiotic resistance and appropriate use of antibiotics, which are the primary risk factors for the development of antibiotic resistance as outlined by the World Health Organisation [66].
The sub-group analysis based on the types of specimens shows that the blood culture had the highest prevalence of MDR Klebsiella spp., at 96% (92-100%), while the other specimen cultures (stool, ear discharge, and vaginal discharge) had the lowest prevalence, at 51% (0.0-100).This can be explained by the fact that the latter specimen came from infections acquired in the community, while the former came from infections acquired in hospitals.Unless multiple blood culture bottles are used to rule out infection versus contamination [67], the other possibility may be related to the risk of contamination of blood culture from the environment by Klebsiella species from hospital-acquired infections.Some microbiology laboratories in developing countries use single bottles for pediatric blood cultures due to supply shortages [68].
Every individual, at any stage of life, is susceptible to AMR.Individuals who are undergoing medical treatment or have compromised immune systems are frequently more vulnerable to infection [69].According to global estimates for 2019, children bear a disproportionate share of the burden of death: of the 1.27 million deaths directly attributable to AMR, 254,000 occurred among those under the age of five, accounting for approximately 20% of all deaths.This is the equivalent of one child passing away almost every two minutes.More than 99 percent of those 254,000 kids are from low-and middle-income countries, and more than half of them pass away in their first month of life.Stated differently, 900 children in high-income countries (HICs) and nearly 253,000 in low-and middle-income countries (LMICs) died directly from AMR [70].Our study also encompassed a subgroup analysis based on the age of the study subjects, which revealed that the lower age group (children and neonates) had a higher prevalence of MDR Klebsiella species (97% (91-100%) vs 96% (92-100%)).Several facets of this growing problem are unique to children.Without immediate action, we are at risk of entering a postantibiotic era in which common infections could once again be fatal in such a rapidly changing environment.This is especially true for children and babies less than a week old, as their gastrointestinal tracts contain populations of MDR bacteria, most likely because of exposure to mother and environmental bacteria during and right after delivery [71].
In our review, a higher MDR Klebsiella species were identified from HAI 97% (97-100%).This might be because staying in a hospital or other healthcare facility increases the risk of contracting an antibiotic-resistant infection.Antibiotic exposure is frequent for patients in these facilities, and they are often prone to a lot of hands-on care which may expose them to resistant bacteria from either medical equipment or health professionals.Furthermore, hospitals are more likely than communities to harbour the majority of resistant bacteria [72].
In general, our study has the following limitations.One of the study's limitations is that it only looked at disc diffusion; it did not find a study which used minimum inhibitory concentration (MIC) or molecular techniques.It only used patient specimens, focused on studies conducted between 2018 and 2022, and failed to determine whether certain specimens, such as urine, faeces, sputum, and discharges, were from infection or colonization.Our study considered the report of MDR Klebsiella spp. as one of the eligibility criteria but not the definition of MDR because the studies used different definitions and some included simply the MDR value.

Conclusion
Our study revealed that the prevalence of MDR Klebsiella species was high in Ethiopia.The subgroup analyses elaborated on the proportion of MDR Klebsiella spp. that was different between regional states, types of specimens, study participants' ages, sources and types of infection.Therefore, integrated action should be taken to reduce the rates of multi-drug-resistant Klebsiella to regional states, age, and focus on clinical features of patients.Standard precautions should be applied to reduce the transmission of MDR Klebsiella spp in hospital and out of the hospital.

Table 3 . Shows the meta-regression based on regional states, types of specimens, study participants' age, source of infection, site of infection and publication year.
https://doi.org/10.1371/journal.pone.0297407.t003